Major Phosphorylation Site (Ser55) of Neurofilament L by Cyclic AMP‐Dependent Protein Kinase in Rat Primary Neuronal Culture
Major Phosphorylation Site (Ser55) of Neurofilament L by Cyclic AMP‐Dependent Protein Kinase in Rat Primary Neuronal Culture
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DOI：
10.1046/j.1471-4159.2000.0740949.x
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发表时间：
2000-03
影响因子：
4.7
通讯作者：
Yu Nakamura;R. Hashimoto;Y. Kashiwagi;S. Aimoto;Eriko Fukusho;N. Matsumoto;T. Kudo;M. Takeda
中科院分区：
文献类型：
--
作者：
Yu Nakamura;R. Hashimoto;Y. Kashiwagi;S. Aimoto;Eriko Fukusho;N. Matsumoto;T. Kudo;M. Takeda
摘要：据报道，神经丝L （NF‐L）的Ser55在神经元中被部分磷酸化，并被环AMP依赖性蛋白激酶（PKA）磷酸化。牛NF - L在低浓度MgCl2 （0.3 mM）中被PKA磷酸化，并被胰蛋白酶消化。胰蛋白酶消化片段通过MALDI/TOF（基质辅助激光解吸和电离/飞行时间）质谱法进行鉴定。在头部区域发现了Ser41， Ser55和Ser62的磷酸化位点，其中Ser55被认为是首选位点。抗Ser55位点特异性磷酸化依赖抗体使NF - L在原代培养的大鼠神经元中检测到Ser55位点磷酸化。20 nM冈田酸处理1小时可提高Ser55的磷酸化水平，10 μM福斯克林共处理1小时可提高Ser55的磷酸化水平。然而，单独使用福斯克林并不能提高磷酸化水平。因此，NF - L在体内可被PKA或PKA样激酶磷酸化Ser55位点；然而，Ser55的磷酸化水平可能受到某些对冈田酸敏感的磷酸酶的调节。
Abstract: Ser55 of neurofilament L (NF‐L) is reported to be partly phosphorylated in neurons and to be phosphorylated by cyclic AMP‐dependent protein kinase (PKA). Bovine NF‐L was phosphorylated by PKA in a low concentration of MgCl2 (0.3 mM) and digested by trypsin. Trypsin‐digested fragments were assigned by MALDI/TOF (matrix‐assisted laser desorption and ionization/time‐of‐flight) mass spectrometry. Phosphorylation sites were found at Ser41, Ser55, and Ser62 in the head region, with Ser55 considered the preferred site. A site‐specific phosphorylation‐dependent antibody against Ser55 rendered NF‐L phosphorylated at Ser55 detectable in primary cultured rat neurons. One‐hour treatment with 20 nM okadaic acid increased the phosphorylation level of Ser55, and co‐treatment with 10 μM forskolin enhanced it. However, forskolin alone did not elevate the phosphorylation level. As a consequence, NF‐L may be phosphorylated at Ser55 by PKA or by a PKA‐like kinase in vivo; however, the phosphorylation level of Ser55 may be modulated by certain phosphatases sensitive to okadaic acid.